J. Biochem. 102, 83-92 (1987)

Occurrence of as a Major in WHHL Rabbit Serum Lipoproteins1

Atsushi HARA and Tamotsu TAKETOMI

Department of Biochemistry, Institute of Cardiovascular Disease , Shinshu University School of Medicine, Matsumoto , Nagano 390

Received for publication, February 12, 1987

Glycosphingolipids in serum and lipoproteins from Watanabe hereditable hyper

li pidemic rabbit (WHHL rabbit), which is an animal model for human familial hypercholesterolemia (FH), were analyzed for the first time in this study . Chylo microns and very low density, low density, and high density lipoproteins contained sulfatide as a major glycosphingolipid (12nmol/ƒÊmol total phospholipids (PL) in chylomicrons, 19nmol/ƒÊmol PL in VLDL, 18nmol/ƒÊmol PL in LDL, and 14nmol/ƒÊ mol PL in HDL) with other minor such as glucosylceramide,

, GM3 , , and globotriaosylceramide. The concentration of sulfatide as a major glycosphingolipid in WHHL rabbit serum (121nmol/ml) was much higher than that in normal rabbit serum (3nmol/ml). Fatty acids of the comprised mainly nonhydroxy fatty acids (C22, 23, and 24) and significant amounts of hydroxy fatty acids (about 10%), whereas long chain bases of the sulfatides comprised mostly (4E)-sphingenine with a significant amount of 4D-hydroxysphinganine (about 10%). Furthermore, sulfatides in the liver and small intestine from normal and WHHL rabbits (where serum lipoproteins are

produced) were determined to amount to 260nmol/g liver in WHHL rabbit, 104 nmol/g liver in control rabbit, 99.6nmol/g small intestine in WHHL rabbit, and 31.2nmol/g small intestine in control rabbit. portions of the sulfatides in the liver were mainly composed of (4E)-sphingenine and nonhydroxy fatty acids, while those in the small intestine were mainly composed of 4D-hydroxysphinganine and hydroxy fatty acids. These results indicated that the sulfatides of serum lipo

proteins were mostly derived from the liver (90% of the total), and that the remaining sulfatides (10% of the total) might be derived from the small intestine. These two sulfatides, which have different ceramide portions, could be useful markers for metabolic and biosynthetic studies of various lipoproteins in WHHL rabbit, and thus would be helpful to further elucidate the relationship between hypercholesterol emia and atherosclerosis in the rabbit.

1 This work was supported in part by Grant-in-Aid for Scientific Research (No. 61480126) from the Ministry of Education, Science and Culture of Japan.

Vol. 102, No. 1, 1987 83 84 A. HARA and T. TAKETOMI

Glucosylceramide, galactosylceramide, lactosylcer was determined by gas-liquid chromatography (9). amide, globotriaosylceramide, globotetraosylcer Phosphorus contents of lipoproteins were deter- amide, and GM3-ganglioside have been found in mined by the method of Bartlett (10). As already human plasma and its lipoproteins (1-3). There reported by the authors (11), aliquots of various are some reports on increased levels of glyco lipoproteins were negatively stained with phos

in serum; for example, an increase photungstic acid (pH 7.2) and dried on collodion of glucosylceramide in Gaucher patients and an carbon grids for electron-microscopic observation

increase of globotriaosylceramide in Fabry patients (Hitachi, model Hu-IIA). These aliquots were (4). It was also reported that the levels of plasma also subjected to electrophoresis on Universal glycosphingolipids were elevated in patients with electrophoresis film (agarose) (Corning Medical, familial hypercholesterolemia (FH) (5, 6), but the U.S.A.). The lipoproteins were stained with Fat mechanism of the increase in glycosphingolipids red 7B or Coomassie brilliant blue R-250. Sodium remains to be solved. It is known that the WHHL dodecyl sulfate polyacrylamide gel electrophoresis rabbit is devoid of LDL-receptor, as is the case was performed according to Laemmli (12). The

in FH. In the present study, we have analyzed procedures for extraction and isolation of glyco glycosphingolipids in whole serum or lipoproteins sphingolipids were described in detail elsewhere in WHHL rabbit in order to further understand (9, 13). Briefly, were extracted from each the glycosphingolipid metabolism in FH. Al lipoprotein fraction, whole serum, liver, or small though Coles and Foote have reported that plasma intestine with a chloroform-methanol mixture. lipoproteins in rabbit contained , di After alkaline treatment to cleave ester lipids, glycosylceramide, triglycosylceramide, and globo sphingolipids were separated into neutral sphin side (7), more detailed information about glyco golipids and acidic sphingolipids with DEAE- sphingolipids of plasma lipoproteins is needed to Sephadex A-25 (acetate form) as described by elucidate the glycosphingolipid metabolism in lipo Ledeen et al. (14). The neutral glycosphingolipids proteins. In this paper, it is reported for the first were acetylated and separated into glycosphingo time that the sulfatide content is highest in the lipids and by silica gel column glycosphingolipid constituents in the serum lipo chromatography and the glycosphingolipids were protein of WHHL rabbit, although the sulfatide finally deacetylated. The neutral glycosphingo is seldom a major glycosphingolipid in other tis- lipids thus obtained were separated from each sues or fluids. other by preparative thin-layer chromatography

(TLC) on plates precoated with Silica gel 60 MATERIALS AND METHODS (Merck, B.R.D.), developed with chloroform- methanol-water (65 : 25 : 4, v/v). The acidic lipids Materials-WHHL rabbits (Japanese white were purified by silica gel column chromatography rabbit) were kindly supplied by Dr. Yoshio Wata as follows; stepwise elution with chloroform- nabe, Kobe University School of Medicine, Kobe. methanol mixtures (95:5, 90:10, 85:15, 80:20, Normal rabbits (Japanese white rabbit) were used and 50:50) gave free fatty acids, sulfatide, and as controls. Blood samples were drawn from the . Sulfatide was further purified by animals after overnight fasting and separated into preparative TLC as above. Analytical procedures sera and erythrocytes by centrifugation. The used for the present study were described in detail pooled sera were used for experiments as well as elsewhere (9, 13). Briefly, after methanolysis of fresh tissues of rabbit liver and small intestine, glycosphingolipid, the resulting fatty acid methyl including mucous membrane. esters were analyzed by gas liquid chromatography Methods-The pooled WHHL rabbit sera (GLC) using an OV-101 silicone capillary column were fractionated into chylomicrons (CM, d<0.93 (0.2 mm•~25m) at 250•Ž. The methyl glycosides g/ml), very low density lipoprotein (VLDL, 1.01 > from the methanolyzate were re-N-acetylated and d> 0.93), low density lipoprotein (LDL, 1.06 > d > analyzed as trimethylsilyl (TMS) derivatives by 1.01), and high density lipoprotein (HDL, 1.15> GLC on an OV-101 silicone capillary column. d> 1.08) by the method of Hatch and Lees (8). The column temperature was programmed from Cholesterol content in each lipoprotein fraction 190 to 260•Ž at 2•Ž/min. Mannitol was used as

J. Biochem. SULFATIDE IN SERUM LIPOPROTEINS 85 an internal standard. Long chain bases were lipoproteins were determined by electronmicro prepared as before (13), and analyzed as TMS- scopic examination. As shown in Fig. 1, the derivatives by GLC on an OV-101 silicone capillary diameters of LDL and VLDL were 17-20 nm and column at 250•Ž. The amount of ganglioside was 33-58 nm, respectively. These lipoproteins were determined with resorcinol reagent (15) or quan also confirmed by electrophoresis on agarose film titated by TLC-densitometry at 570 nm (16). (data not shown). Simple lipid compositions of VLDL and LDL were determined by GLC anal

RESULTS ysis of cholesterol or fatty acid methyl ester. VLDL contained 54% cholesterol ester, 35% tri Fractionation of Various Lipoproteins•\The glyceride, and 11% free cholesterol, while LDL pooled sera of fasting WHHL rabbits were frac contained 62% cholesterol ester, 23% triglyceride, tionated into CM, VLDL, LDL, and HDL by the and 15 % free cholesterol.

procedure of Hatch and Lees (8). The lipoprotein Glycosphingolipids in Serum, and CM, VLDL, composition of the WHHL rabbit sera was deter- LDL, and HDL-Crude sphingolipids were sepa mined on the basis of cholesterol content as fol- rated into neutral and acidic sphingolipids on lows: CM (1.9%), VLDL (24.5%), LDL (71.4%), DEAE-Sephadex A-25. After sphingomyelin was and HDL (2.2 %). The size and shape of these removed from the neutral , relatively

Fig. 1. Electron micrographs of VLDL and LDL. Each lipoprotein was negatively stained with phosphotungstic acid and dried on a collodion carbon grid.

Vol. 102, No. 1, 1987 86 A. HARA and T. TAKETOMI

small amounts of neutral glycosphingolipids were major glycosphingolipid revealed that galactose obtained. They were separated from each other was the only component. The IR spectrum of by preparative TLC and identified by GLC anal the glycosphingolipid was similar to that of sul ysis of TMS derivatives of the methylglycosides. fatide: 2,920 and 2,850cm-1 due to CH2 and The neutral glycosphingolipids were mostly com CH3 groups, 1,640 and 1,550cm-1 due to amide posed of glucosylceramide, galactosylceramide, linkage, 1,460cm-1 due to CH, and CH groups, lactosylceramide, globotriaosylceramide, and other 1,240cm-1 due to SO,H group and 1,000-1,100 minor oligoglycosylceramides such as globotetra cm-1 due to OH group. Periodate oxidation with osylceramide (tentatively identified by TLC and 0.2M sodium periodate (18) and solvolysis with GLC analysis of TMS derivatives of the methyl 0.1N HCl in methanol (19) were performed, and glycosides). On the other hand, relatively large the results are shown in Fig. 3. The glycosphingo amounts of acidic glycosphingolipids were also lipid which had the same Rf value as sulfatide separated from each other by TLC (Fig. 2). One was stable to periodate oxidation, whereas it was major band which has the same Rf value as sul converted to galactosylceramide by solvolysis. fatide was detected in all the lipoprotein fractions Galactosylceramide obtained by solvolysis had the and in whole sera of WHHL rabbit. The bands same R f value as nonhydroxy fatty acid-containing located between sulfatide and GM3 in lanes B-3 galactosylceramide on TLC. These results con- and B-4 were negative to the anthrone reagent, firmed that the structure of the glycosphingolipid indicating nonglycolipid nature. The bands which was identical with sulfatide. Ganglioside contents migrated slightly faster than the major band in and compositions were similar in each lipoprotein each lane were methanolyzed and analyzed by fraction. In the case of LDL, GM3 (51% of the GLC. No sugars were found in the methano total sialic acid) and GD3 (26%) were the major lyzates, but only cholesterol. The mobility of the gangliosides in addition to small amounts of GD1a band was identical to that of cholesterol sulfate. Thus, the band was tentatively identified as cho lesterol sulfate. GLC analysis of sugar in the

Fig. 2. Thin-layer chromatograms of acidic glyco sphingolipids of serum, and CM, VLDL, LDL, and Fig. 3. Solvolysis and periodate oxidation of sulfatide. HDL. The plates were developed with chloroform- The plates were developed with chloroform-methanol- methanol-water (65:25:4, v/v). The bands were water (65 : 25 : 4, v/v). The bands were detected as detected with cupric-phosphoric acid charring reagent in Fig. 2. 1, galactosylceramide from porcine spinal (17). A-1, GM3 (control); A-2, sulfatide from porcine cord (control); 2, galactosylceramide treated with perio spinal cord (control); A-3, CM; A-4, VLDL; A-5, date (control); 3, sulfatide treated with periodate; 4 LDL; A-6, HDL. B-1, sulfatide from porcine spinal and 5, sulfatide from LDL; 6, sulfatide from porcine cord (control); B-2, GM3 (control); B-3, normal rabbit spinal cord (control); 7, solvolysate of sulfatide; 8, serum; B-4, WHHL rabbit serum. galactosylceramide from porcine spinal cord (control).

J. Biochem. SULFATIDE IN SERUM LIPOPROTEINS 87

(7%), GT1b (5%), GQlb (1%), and unknown the major apolipoprotein of LDL. It should be ganglioside (8%). Table I summarizes the glyco noted that the sugar contents of glycosphingolipids sphingolipid contents in whole serum, and CM, were relatively lower than that of apolipoprotein B. VLDL, LDL, and HDL . As shown in Table I, Fatty acid compositions of glycosphingolipids WHHL rabbit serum contained much larger are shown in Table II. Hydroxy fatty acids were amounts of glycosphingolipids than normal rabbit detected in glucosylceramide, galactosylceramide, serum. It was found that sulfatides were pre and sulfatide. Major fatty acids of these glyco dominant among the glycosphingolipid constitu sphingolipids were C22, C23, and C24. Lacto ents, and the sulfatide contents were at almost the sylceramide and globotriaosylceramide contained same levels in all the lipoproteins of WHHL rabbit only nonhydroxy fatty acids such as C22, C23, sera when the values were expressed based on the and C24 as major fatty acids. Although GM3

phospholipid content of each lipoprotein. The also contained only nonhydroxy fatty acids, the glycosphingolipid composition in normal rabbit major fatty acids were C18 and C22. sera seemed to be similar to that of WHHL rabbit The long chain base composition of sulfatide sera except for glucosylceramide content, which from LDL was analyzed by GLC as TMS deriva was comparable to sulfatide content in the normal tives. The sulfatide contained (4E)-sphingenine rabbit sera. (77%), sphinganine (9%), 4D-hydroxysphinganine The sugar content in the proteinaceous residue (12 %), and 4D-hydroxyicosasphinganine (2 %). of delipidated LDL of WHHL rabbit sera was The long chain base composition and fatty estimated by GLC of TMS derivatives of the acid composition of galactosylceramide, a metabo methyl glycosides, and the values expressed based lite of sulfatide, were investigated. Figure 4 shows on the phospholipid contents of LDL were as the result of TLC analysis of glucosylceramide and follows: N-acetylglucosamine (103nmol/ƒÊmol PL galactosylceramide from LDL using 3 % Borax- in LDL), mannose (86.6nmol/ƒÊmol PL in LDL), impregnated silica gel plates. The mixture of sialic acid (55.0nmol/ƒÊmol PL in LDL), galactose glucosylceramide and galactosylceramide was sepa rated into four different bands; from band A to (44.1nmol/ƒÊmol PL in LDL), N-acetylgalacto samine (5.14nmol/ƒÊmol PL in LDL), and glucose band D in Fig. 4. Sugar, fatty acids, and long chain bases of each glycosphingolipid were deter- (4.31nmol/ƒÊmol PL in LDL). These sugars may be the components of apolipoprotein B, which is mined by GLC. As shown in Table III, band A

TABLE I. Glycosphingolipid contents in whole serum, and CM, VLDL, LDL, and HDL of WHHL rabbit.

a Total phospholipids. b Not determined. c WHHL rabbit serum contained 520±55 mg total phospholipids/dl.

d This value is equivalent to 18.0nmol/ƒÊmol PL.

Vol. 102, No. 1, 1987 88 A. HARA and T. TAKETOMI

TABLE II. Fatty acid compositions of glycosphingolipids in various lipoproteins of WHHL rabbit (%).

a Trace . b Hydroxy fatty acid.

Fig. 4. Borax-impregnated thin-layer chromatogram of glucosylceramide and galactosylceramide obtained Fig. 5. Thin-layer chromatogram of sulfatides from from LDL of WHHL rabbit. The plate was developed normal liver and small intestine. The plate was devel with chloroform-methanol-3.5% ammonia (60:35: oped with chloroform-methanol-water (65 : 25 : 4, v/ 8, v/v). The bands were detected as in Fig. 2. 1, v). The bands were detected as in Fig. 2. 1 and 4, glucosylceramide from Gaucher spleen; 2, galactosyl ceramide from porcine spinal cord; 3, glucosylceramide sulfatide from porcine spinal cord; 2, sulfatide fraction and galactosylceramide from LDL. from liver; 3, sulfatide fraction from small intestine.

J. Biochem. SULFATIDE IN SERUM LIPOPROTEINS 89

TABLE III. Fatty acid and long chain base compositions of glucosylceramides and in LDL of WHHL rabbit (%).

a Trace . b Hydroxy fatty acid.

(which accounted for 68% of glucosylceramide) (59.0 g of wet tissue) or WHHL rabbit small contained nonhydroxy fatty acids and dihydroxy intestine (15.5 g of wet tissue) was subjected to bases. Band B (which accounted for 32% of silica gel column chromatography (Silica gel 60, glucosylceramide) contained nonhydroxy or hy Merck, 10 x 60 mm). Free fatty acids were eluted droxy fatty acids and trihydroxy bases. Band C with chloroform-methanol (95:5, 40ml) and (90: (which accounted for 74% of galactosylceramide) 10, 10ml), then sulfatide was eluted with chloro contained nonhydroxy or hydroxy fatty acids and form-methanol (90:10, 10ml, 85:15, 20ml, and dihydroxy bases. Finally, band D (which ac- 80:20, 175ml). The sulfatide fraction thus ob counted for 26% of galactosylceramide) contained tained from normal rabbit tissues was analyzed nonhydroxy or hydroxy fatty acids and trihydroxy by TLC as shown in Fig. 5. The extract from the bases. The doublets of band C and band D are liver showed one major band, while that from the due to the differences of fatty acids. Glucosyl small intestine showed three bands. The band ceramide and galactosylceramide contained C22, which had a lower R f value than that of sulfatide C23, and C24 as major nonhydroxy or hydroxy in the liver was negative to the anthrone reagent. fatty acids. Thus, no further analysis of this band was done. Sulfatide in Liver and Small Intestine-Acidic The bottom band of the three major bands in fraction obtained from normal rabbit small intestine was also negative to the anthrone liver (99.5 g of wet tissue), WHHL rabbit liver reagent, indicating non-glycosphingolipid nature. The top band was determined to be cholesterol (14.6 g of wet tissue), normal rabbit small intestine

Vol. 102, No. 1, 1987 90 A. HARA and T. TAKETOMI

sulfate by GLC analysis. Thus, the middle band TABLE IV. Fatty acid compositions of sulfatides in in the small intestine and one major band in the normal and WHHL rabbits (%). liver were identified as sulfatides which corre sponded to that in LDL. The sulfatide contents were determined by GLC analysis of TMS deriva tives of the methylglycosides. The normal and WHHL rabbit livers contained 104 and 260nmol sulfatide/g tissue, respectively, while the normal and WHHL rabbit small intestines contained 31.2 and 99.6nmol sulfatide/g tissue, respectively. It should be noted that the liver and the small intes tine of WHHL rabbit contained larger amounts of sulfatide than normal. The sulfatide contents in the rabbit livers were very high as compared with that in human liver, which contained 1nmol sulfatide/g tissue (20). The fatty acid compositions of the sulfatides from the livers and the small intestines of normal and WHHL rabbits are shown in Table IV. The chain lengths of the major fatty acids of the sulfatides were C22, C23, and C24 in both organs, but the sulfatide in both livers contained mostly nonhydroxy fatty acids, while that in the small intestines contained mostly hy droxy fatty acids. There was no marked differ ence in fatty acid composition of sulfatides be tween normal and WHHL rabbits. Table V shows the long chain base compo sitions of the sulfatides in the livers and the small intestines. Normal and WHHL rabbits showed a similarity in their long chain base compositions. The sulfatides in the livers contained mostly di hydroxy bases, while those in the small intestines contained mostly trihydroxy bases. It was thus noted that the sulfatides in the livers contained ceramide which was mainly composed of nonhy droxy fatty acids and dihydroxy bases, whereas the sulfatides in the small intestines contained a Trace . b Hydroxy fatty acids. ceramide which was mostly composed of hydroxy

TABLE V. Long chain base compositions of sulfatides in normal and WHHL rabbit (%).

J. Biochem. SULFATIDE IN SERUM LIPOPROTEINS 91

fatty acids and trihydroxy bases in both normal obtained from the scanning sodium dodecyl sulfate and WHHL rabbits. polyacrylamide gel electrophoresis of LDL apo lipoprotein at 570 nm at the gel concentration of DISCUSSION 5% after staining with Coomassie brilliant blue R-250 (data not sown). The content of apo There have been no reports on the presence of lipoprotein B-100, which might be derived from sulfatides in human and other animal sera or the liver, was approximately 90%, while that of lipoproteins. It was found for the first time in apolipoprotein B-48, which might be derived from the present study that sulfatides are the major small intestine, was about 10%. High contents glycosphingolipid in all the lipoprotein fractions of sulfatides in lipoproteins as well as different such as CM, VLDL, LDL, and HDL in WHHL ceramide portions of sulfatides derived from the rabbit serum. Other minor glycosphingolipids such two different sources suggest that sulfatides may as glucosylceramide, galactosylceramide, GM3 serve as useful tools in studying lipoprotein metab ganglioside, lactosylceramide, globotriaosylcer olism and biosynthesis in WHHL rabbits, and thus amide, and other oligohexosylceramides were also be helpful to further investigate the relationship detected in the lipoproteins, as has already been between hypercholesterolemia and atherosclerosis reported in human sera (1-3). The content and in WHHL rabbits. Wago (22) has already re- fatty acid composition of sulfatides were rather ported that intravenous administration of sulfatide similar in each lipoprotein fraction. These find to cholesterol-fed rabbits inhibited the elevation of ings not only support the metabolic conversion of serum total lipid and total cholesterol, and miti VLDL to LDL via IDL, but also indicate the gated the atheromatous involvements in the aorta. possibility that sulfatides are freely exchangeable Also, Roberts et al. (23) have recently re- among lipoproteins like neutral glycosphingolipids ported very interesting data concerning highly (21). It is well known that lipoproteins are bio- specific interactions between sulfatides and cell synthesized independently in both liver and small adhesion proteins such as laminin, thrombospondin intestine. Thus, in order to examine whether the and von Willebrand factor. The presence of sul liver and the small intestine contain sulfatides fatides in serum lipoproteins suggests that the which are found in the lipoproteins, these tissues sulfatide-containing lipoproteins may play a role were subjected to analyses of glycosphingolipids. in regulating the physiological function of the cell If the sulfatides in these organs can be distinguished adhesion proteins. However, it remains to be in terms of fatty acid and long chain base com- investigated whether sulfatides exist in the sera position, the origins of the lipoproteins may be or serum lipoproteins of all mammalian species. deduced from the sulfatides compositions. From the contents of hydroxy fatty acids of the sulfatides REFERENCES in the WHHL rabbit liver (HFA content: 3% of 1. Svennerholm, E. & Svennerholm, L. (1963) Nature total fatty acids) and in the WHHL rabbit small 198,688-689 intestine (HFA content: 87% of total fatty acids), 2. Vance, D.E. & Sweeley, C.C. (1967) J. Lipid Res. 88% of the sulfatides in LDL (HFA content: 13 8, 621-630 of total fatty acids) were assumed to be derived 3. Yu, R.K. & Ledeen, R.W. (1972) J. Lipid Res. 13, from the liver, and the remaining sulfatides (12%) 680-686 from the small intestine. On the other hand, 4. Vance, D.E., Krivit, W., & Sweeley, C.C. (1969) when trihydroxy bases of the sulfatides in WHHL J. Lipid Res. 10, 188-192 5. Chatterjee, S. & Kwiterovich, P.O. (1976) Lipids rabbit were used for calculation in the same way 11,462-466 (Table V), 89 % of the sulfatides in LDL were 6. Dawson, G., Kruski, A.W., & Scanu, A.M. (1976) assumed to be derived from the liver, and 11% J. Lipid Res. 17, 125-131 from the small intestine. Thus, it was suggested 7. Coles, E. & Foote, J.L. (1974) J. Lipid Res. 15, that the sulfatides in serum lipoprotein of WHHL 192-199 rabbit were mostly derived from the liver (90%) 8. Hatch, F.T. & Lees, R.S. (1968) in Advances in and, to a lesser extent, from the small intestine Lipid Research (Paoletti, R. & Kritchevsky, D., (10%). This ratio was also supported by the data eds.) Vol.6, pp. 1-68, Academic Press, New York

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